PROJECT SUMMARY The ubiquitin proteasome system (UPS) regulates a non-lysosomal intracellular protein degradation pathway that is required for cell-cycle progression, signal transduction, removal of damaged proteins, and maintenance of normal protein homeostasis. Dysfunction of the UPS is associated with pathogenesis of various human diseases including multiple myeloma (MM); therefore, inhibitors of UPS pathways offer great promise as a novel therapeutic strategy. We have characterized targeting of UPS in MM using our in vitro and in vivo models of the MM cell in the bone marrow (BM) milieu, specifically delineating the molecular and cellular mechanisms whereby proteasome inhibitors target tumor cells, host tumor interactions, and the BM microenvironment to overcome drug resistance. These preclinical studies and clinical trials provided the basis for FDA approval of proteasome inhibitors (PI) bortezomib, carfilzomib, and ixazomib for the treatment of relapsed/refractory and newly diagnosed MM, and importantly, validated the 20S proteasome in the UPS as a therapeutic target in MM. Even though these therapies are major advances, they are associated with possible off-target toxicities and the eventual development of drug-resistance. Therefore, our more recent studies have focused on targeting enzymes modulating protein ubiquitin-conjugation and -deconjugation rather than the proteasome itself, with the goal of generating more specific and less toxic anti-tumor agents. In the last grant period, we advanced inhibitors targeting deubiquitylating enzymes USP14/UCHL5 from the bench to the bedside and first in man clinical trials. Our most recent efforts have focused on targeting another major component of UPS, Ubiquitin Receptors (UbRs). Our Preliminary Studies show that UbR Rpn13/ADRM1 is more highly expressed in MM cells than in normal plasma cells, and that inhibiting Rpn13 triggers MM cell growth inhibition, even in MM PI resistant MM. The current proposal aims to investigate the hypothesis that inhibition of the UPS at the level of UbRs, upstream of the proteasome, can inhibit MM cell growth and overcome PI resistance. To achieve these goals, we will pursue the following Specific Aims: Specific Aim 1: Functional characterization of Ubiquitin Receptors in MM pathogenesis. Specific Aim 2: To design specific UbR inhibitors and/or degraders, and assess their in vitro specificity and mechanism of action, either alone or in scientifically-informed combinations. Specific Aim 3: To pre-clinically evaluate in vivo anti-MM activity of novel UbR inhibitors and/or degraders, either alone or in combination therapies, for translation into clinical trials. This new paradigm to target UPS pathways in MM at the level of Ubiquitin Receptors (UbRs) has great promise not only to overcome PI resistance and improve patient outcome, but may also serve as a model for targeted therapeutics in other cancers.